The present invention relates to a cell surface antigen determination method for determining an immunocyte having an immune antigen-antibody complex composed of an antigen and an antibody combined therewith in a liquid which contains immunocytes having various antigens and also containing many kinds of antibodies. The invention also relates to an apparatus used in this method.
The following are general conventional methods for immune reaction:
1. Qualitative techniques such as a ring test, a capillary test, a tube test, an immune diffusion test, and quantitative techniques such as a quantitative precipitation method, a laser nephelometry, and a quantitative immune diffusion method. PA1 2. Hemolysis and bacteriolysis assays. PA1 3. Agglutination techniques such as a cell agglutination test, an anti-globulin test (Coombs test), a passive hemagglutination method (PHA, HA), and an immune adherence method (IA). PA1 4. Passive cutaneous anaphylaxis assay (PCA). PA1 5. Labeled antibody techniques such as fluoroimmunoassay (FIA), enzyme immunoassay (EIA), and radio immunoassay (RIA). PA1 a container for holding a first liquid containing an immunocyte having at its surface an immune antigen-antibody complex composed of an antigen and an antibody combined with the antigen; PA1 a substance secured to part of an inner surface of the container, the substance being capable of specifically combining with immunoglobulin; PA1 a second liquid contained in the container together with the first liquid, the second liquid being higher in specific gravity than the first liquid; and PA1 container rotating means for rotating the container in such a manner that the part of the inner surface is disposed most remote from the axis of rotation.
The above methods have been improved in various ways depending on their applications, and automation and labor-saving techniques have been achieved with respect to these methods. However, in a general immune reaction, in the absence of a monoclonal antibody of a highly specific nature or a complement, or even in the presence of a complement, the complement activity in many cases is not efficient. In such a case, a background increase due to non-specific reactions and other factors cannot be disregarded, and as a result it is very difficult to determine an intended specific reaction.
For example, the following problems are encountered with a lymphocyte cytotoxicity test (LCT) of a human leukocyte antigen system A (HLA) based on hemolysis and bacteriolysis assays. In this test, an antiserum which is derived from a multipara and has various specificities and activities is used as an antibody, and this antibody is reacted with a lymphocyte HLA antigen to determine the HLA type. In this case, a complement, which is specifically activated in cytotoxicity under the influence of an immune antigen-antibody complex produced as a result of the reaction, and a coloring matter for dyeing destroyed lymphocytes are used. The degree of specificity of the lymphocyte is evaluated from the color matter-dyeing frequency proportional to the immunocyte cytotoxicity frequency. However, when the antigen concentration is low, for example, in the case where the antigen is diffused at the surface of one lymphocyte, the complement activity is not sufficient. Also, in IgG4 sub-class antibody reactions, complement activity is not found. In such a case, it is very difficult to determine the specificity of the lymphocyte. Further, although more than 140 kinds of HLA antigens have been discovered, much larger numbers are believed to exist. However, no determination method has yet been established.